Candidiasis: a novel molecular differential diagnosis through real-time PCR followed by high-resolution thermal denaturation

Invasive infections caused by Candida species, especially in immunocompromised patients, are a growing global health concern due to high mortality rates and increasing antifungal resistance. Rapid and accurate identification of the etiological agent is essential for timely and appropriate treatment. In this study, we developed a one-hour multiplex real-time PCR assay coupled with high-resolution melting (qPCR-HRM) for the simultaneous identification of Candida albicans, Candida parapsilosis and Candida auris, using five specifically designed primers and the intercalary dye Syto9. The assay demonstrated species-specific melting profiles: 80.6 °C for C. albicans, 84.3 °C for C. parapsilosis, and 82.9 °C for C. auris, with no cross-reactivity except among species within the C. parapsilosis complex. Analytical sensitivity was evaluated using simulated blood cultures and achieved detection limits of 1000, 200, and 20 CFU/PCR for C. albicans, C. parapsilosis, and C. auris, respectively. This assay is a cost-effective, rapid, and specific method for early detection of key Candida species in bloodstream infections, ideal for clinical laboratories in resource-limited settings.